Preparation Of Polyphenols And β-lactoglobulin Complexes And Their Mechanism Of Inhibiting Xanthine Oxidase

Xanthine Oxidase（XOD） is a key enzyme in the body to metabolize hypoxanthine and xanthine to produce uric acid.Polyphenol monomers derived from natural products have good biological activity and safety,can inhibit xanthine oxidase,reduce the synthesis of uric acid and relieve gout symptoms.Therefore,it is important to screen out monomers with a good XOD inhibition effect from polyphenol monomers,and develop a highly safe compound that can combine multiple polyphenol monomers at the same time,which is of great significance to the symptom relief of gout patients.β-lactoglobulin（β-LG）is widely used as a carrier for active substances and can resist pepsin digestion in the human body,so polyphenol monomers can be smoothly transported to the intestine to be absorbed by the small intestine by combining withβ-LG,and then release the polyphenol to exert its active effect.In this study,the polyphenol monomers Resveratrol（RES）,epigallocatechin gallate（EGCG）and hesperidin（HES）were the main research objects,andβ-lactoglobulin was selected.β-lactoglobulin was chosen as the carrier for ligand binding,the inhibition kinetics,inhibition effect and mechanism of polyphenol monomer on XOD,the binding mechanism of polyphenol andβ-LG,and the inhibitory effect ofβ-LG-tri-ligand compound on XOD were studied.The main research methods and results are as follows:（1）First,the ultraviolet spectrophotometry was used to compare the inhibitory effects of six common and highly bioactive polyphenol monomers on XOD,and finally three polyphenols were selected-EGCG,resveratrol and hesperidin.The inhibition kinetics of XOD was calculated,and the mechanism of binding of polyphenol monomers to XOD was simulated by fluorescence quenching experiment combined with molecular docking.The EGCG,resveratrol and hesperidin half-inhibited concentrations(IC₅₀)at room temperature were 68.99μg/m L、59.62μg/m L and 59.27μg/m L,Lineweaver-Burk double reciprocal mapping shows that they are competitive inhibitors of xanthine oxidase.The combination of EGCG,resveratrol and hesperidin and XOD leads to quenching the endogenous fluorescence of the enzyme,the quenching mechanism is static quenching,and the three polyphenols can bind to the amino acid residues of XOD and inhibit the activity of xanthine oxidase.（2）Molecular fluorescence,Fourier infrared,circular dichroism,ultra-high performance liquid chromatography,and molecular docking simulations were used to reveal the binding mechanism ofβ-LG and polyphenol monomers.The results of molecular fluorescence spectroscopy indicate that EGCG,resveratrol and hesperidin quench the protein fluorescence through a static mechanism.The results of Fourier infrared spectroscopy and circular dichroism show that the polyphenol monomer can change the secondary structure ofβ-LG by increasing theβ-sheet and decreasing theβ-turn structure.The content of regular curl basically does not change much.Ultra-high performance liquid chromatography results show that polyphenol monomers can interact withβ-LG to formβ-LG-single ligand complexes which is stable at room temperature and heating conditions.Molecular docking simulation results confirmed that EGCG,resveratrol and hesperidin mainly stably bind to the central hydrophobic cavity and the hydrophobic surface ofβ-LG through hydrogen bonding and hydrophobic interaction.（3）The effect of adding polyphenol monomer on the fluorescence spectrum ofβ-LG-monoligand complexes solution was studied by molecular fluorescence spectrometry,and The order of addition ofβ-LG-tri-ligand complexes was determined to be hesperidin,EGCG,resveratrol.The molecular docking software Auto Dock4.2 and Py MOL simulated the binding mechanism.Finally,the inhibitory effect ofβ-LG-polyphenol complexes on XOD was detected by UV spectrophotometry,and theβ-LG-hesperidin mono-ligand complexes was obtained.The IC₅₀ ofβ-LG-hesperidin-EGCG diligand complexes andβ-LG-hesperidin-EGCG-resveratrol triligand complexes are 132.52μM,68.29μM and41.32μM,respectively.The final inhibitory effect ofβ-LG-tri-ligand complex is more than7 times that of hesperidin monomer,about 11 times that of EGCG monomer,and about 19times that of resveratrol monomer.Because the aboveβ-LG-polyphenol complexes has a good inhibitory effect on XOD,it has a good prospect in the development and utilization of natural active ingredients and the application of uric acid-lowering functional foods.